EP3655421B1 - Procédés de purification de protéines ayant une activité carboxypeptidase de tubuline et leurs inhibiteurs à base peptidique - Google Patents

Procédés de purification de protéines ayant une activité carboxypeptidase de tubuline et leurs inhibiteurs à base peptidique Download PDF

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EP3655421B1
EP3655421B1 EP18743458.4A EP18743458A EP3655421B1 EP 3655421 B1 EP3655421 B1 EP 3655421B1 EP 18743458 A EP18743458 A EP 18743458A EP 3655421 B1 EP3655421 B1 EP 3655421B1
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peptidic
protein
seq
tubulin
amino acid
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EP3655421A1 (fr
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Krzysztof Rogowski
Siem VAN DER LAAN
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Centre National de la Recherche Scientifique CNRS
Universite de Montpellier I
Universite de Montpellier
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Universite de Montpellier I
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    • C—CHEMISTRY; METALLURGY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8103—Exopeptidase (E.C. 3.4.11-19) inhibitors
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
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    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
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    • C12N9/93—Ligases (6)
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17017—Tubulinyl-Tyr carboxypeptidase (3.4.17.17)
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    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • Bosson et al. discloses a method of purification of tubulin comprising its immunoprecipitation. But the present invention distinguishes from this prior art in that the enzyme exhibiting tubulin carboxylase activity has not been purified in this document, nor have been identified and prepare peptidic inhibitors of this enzyme beyond a vague indication that the C-terminal sequence of tubulin can inhibit it. Different methods for enrichment of the enzyme, different inhibitors are proposed in prior art but none would help man skilled in the art to achieve the present invention.
  • the present invention proposes methods for identifying both, the enzymes that possess TCP activity and amino-acid or peptidic based inhibitors as disclosed in the claims 6 to 11 regardless of the original tissue and/or organism.
  • the present invention proposes to purify TCP activity from a biological extract and to use such purified biological extract, which exhibits native TCP activity to test and identify amino-acid or peptidic based inhibitors as disclosed in the claims 6 to 11.
  • the microtubules comprise synthetic microtubules and/or ⁇ -tubulins, with labeled C-terminal Y.
  • the peptidic moiety of the peptidic based inhibitor candidate is constituted of between 2 and 20 amino acids of the most C-terminal amino acids of an alpha-tubulin of SEQ ID N°12.
  • the disorder is preferably selected from neurodegenerative diseases, preferably selected from Alzheimer disease, Parkinson disease, psychiatric disorders, and neural disorders, neuronal regeneration disorders, cancers, preferably selected from colon cancer and neuroblastoma, muscular dystrophies, heart diseases, vascular disorders, infertility, retinal degeneration, and ciliopathies.
  • neurodegenerative diseases preferably selected from Alzheimer disease, Parkinson disease, psychiatric disorders, and neural disorders, neuronal regeneration disorders, cancers, preferably selected from colon cancer and neuroblastoma, muscular dystrophies, heart diseases, vascular disorders, infertility, retinal degeneration, and ciliopathies.
  • the method further comprises a step of selecting proteins that contain a protease domain.
  • proteins that contain a protease domain may be tested.
  • various inhibitors of cysteine, aspartic, serine, threonine proteases and metalloproteases may be tested.
  • the fraction of proteins with a tubulin carboxypeptidase activity is obtained from a brain extract, such as a brain extract from pigs, and the mass spectrometric data are aligned with human reference sequences, in order to identify corresponding human proteins.
  • the fraction of proteins comprises at least one protein selected from the proteins listed in Table 1.
  • the fraction of proteins with a tubulin carboxypeptidase activity is further contacted with microtubules and the level of isolated tyrosine (Y) is measured, thereby confirming the tubulin carboxypeptidase activity of the fraction of proteins.
  • the microtubules comprise synthetic microtubules and/or ⁇ -tubulins, with labeled C-terminal Y.
  • Pharmaceutically acceptable carriers include, for example, aqueous solutions such as water, 5% dextrose, or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters that are suitable for administration to a human or non-human subject.
  • a pharmaceutically acceptable carrier or composition is sterile.
  • a pharmaceutical composition can comprise, in addition to the active agent, physiologically acceptable compounds that act, for example, as bulking agents, fillers, solubilizers, stabilizers, osmotic agents, uptake enhancers, etc.
  • native inherent activity is understood the naturally obtained enzymatic activity contained within the biological sample and which has been obtained solely by described extraction method from a specific tissue, organ of biological sample.
  • native activity corresponds to natural, unadorned or unchanged state; it has not been engineered nor adapted and reflects physiologically present activity in the studied biological sample, such as but not limited to a specific tissue/organ.
  • the isolated MAPs from crude brain extracts were contacted to the radioactively labeled MTs in absence or presence of different peptidic inhibitors or an increasing concentration of peptidic inhibitor. Release of radioactive tyrosine by native TCPase containing brain MAPs was measured by quantification of radioactivity in both the soluble and insoluble fraction of the reaction using a liquid scintillator counter.
  • TCPase inhibitor C2C12 cells were treated with or without EEY peptide ( Figure 12 ).
  • the protein expression of Myosin was monitored by western blotting as control for muscle differentiation. Vinculin acts as loading control. Acetylation and detyrosination of the microtubules was assessed. Whereas acetylation increases during differentiation (Ac-Tubulin) no difference in the status could be observed in the treated cells. Interestingly, detyrosination levels were increased already at day after onset of myogenic differentiation.
  • the presence of the TCPase inhibitor clearly inhibited detyrosination ( ⁇ -1 Tubulin), further supporting the notion that the TCPase inhibitor is cell permeable and acts on intrinsic TCPase activity.
  • the Antibodies recognizing detyrosinated tubulin (deTyr-tub), beta tubulin (E7, hybridoma) and vinculin (Sigma) were used to detect protein levels.
  • a secondary antibody coupled to HRP (Cell Signaling) was used for detection of the protein of interest.
  • Example 7 Study of the detyrosination process of microtubules in neurodegenerative diseases
  • the cells were maintained at the neural progenitor stage and samples were collected every day in a RIPA buffer (50 mM Tris HCl, 150 mM NaCl, 1.0% (v/v) NP-40, 0.5% (w/v) Sodium Deoxycholate, 1.0 mM EDTA, at a pH of 7.4), and quantitation of total protein performed using BCA kit (Thermo Fisher Scientific). A 20 ⁇ g protein sample of a total cell extract was run on 10% SDS-PAGE, transferred to nitrocellulose, and probed with each antibody.
  • RIPA buffer 50 mM Tris HCl, 150 mM NaCl, 1.0% (v/v) NP-40, 0.5% (w/v) Sodium Deoxycholate, 1.0 mM EDTA, at a pH of 7.4
  • BCA kit Thermo Fisher Scientific
  • CHL-1 cells that is a human melanoma cell line and HEK cells that have been demonstrated the ability to form colonies in soft agar and tumors of different size with varying frequencies in immunocompromized mice
  • a peptidic inhibitor to reduced taxol induced detyrosination.
  • Cells were routinely cultured in a standard humidified tissue culture incubator at 37°C in presence of 5% CO2 and plated in a 6-wells culture dish. The cells were treated for 2 hours with 10 ⁇ M Taxol in absence or presence of 50 ⁇ M peptidic inhibitor.

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Claims (13)

  1. Procédé pour purifier des protéines ayant une activité de tubuline carboxypeptidase à partir d'un extrait biologique, comprenant :
    (a) la centrifugation de l'extrait biologique à une température comprise entre 0 et 10°C, de préférence entre 2 et 5°C, mieux encore à 2 °C ;
    (b) la récupération du surnageant de l'étape (a) et la mise en œuvre d'un premier cycle de polymérisation de microtubules par addition de GTP et incubation du mélange à une température comprise entre 35 et 40°C, de préférence à 37 °C ± 2°C, puis centrifugation ;
    (c) la récupération du culot de l'étape (b), la remise en suspension dans du tampon glacé, l'incubation à 4 °C ± 1 °C, et la mise en œuvre d'un deuxième cycle de polymérisation de microtubules par addition de GTP et incubation du mélange à 37 °C ± 2 °C, puis centrifugation ;
    (d) la récupération du culot de l'étape (c), la remise en suspension dans du tampon glacé, l'incubation à 4 °C ± 1 °C, et la mise en œuvre d'un troisième cycle de polymérisation de microtubules par addition de GTP et incubation du mélange à 37 °C ± 2 °C, puis centrifugation ;
    (e) la remise en suspension du culot de l'étape (d) et la soumission du mélange à une chromatographie par échange d'ions et la récupération de l'écoulement traversant ;
    (f) la précipitation des protéines de l'écoulement traversant avec une solution de sulfate d'ammonium saturée à 60 % ;
    (g) la soumission de la fraction précipitée de l'étape (f) à une chromatographie hydrophobe et l'élution par diminution progressive de la concentration de sulfate d'ammonium jusqu'à zéro pour que soit récupérée la fraction de protéines ayant une activité de tubuline carboxypeptidase.
  2. Procédé pour purifier des protéines selon la revendication 1, dans lequel
    - le premier cycle de polymérisation comprend :
    (i) l'addition de GTP et l'incubation du mélange à 37 °C ± 2 °C pendant 30 minutes ± 10 minutes ;
    (ii) la centrifugation à 22 000 g ± 1 000 g à 37 °C ± 2 °C pendant 45 minutes ± 10 minutes ;
    et/ou
    - le deuxième cycle de polymérisation comprend :
    (i) l'incubation du mélange sur de la glace pendant 30 minutes ± 10 minutes ;
    (ii) la centrifugation à 150 000 g ± 10 000 g pendant 30 minutes ± 10 minutes ;
    (iii) la récupération du surnageant et l'addition de GTP ;
    (iv) l'incubation du mélange à 37 °C ± 2 °C pendant 30 minutes ± 10 minutes ;
    (v) la centrifugation à 50 000 g ± 1 000 g à une température comprise entre 30 °C et 37 °C pendant 30 minutes ± 10 minutes ;
    et/ou
    - le troisième cycle de polymérisation comprend :
    (i) l'incubation du mélange sur de la glace pendant 30 minutes ± 10 minutes ;
    (ii) la récupération du surnageant et l'addition de GTP ;
    (iii) l'incubation du mélange à 37 °C ± 2 minutes pendant 30 minutes ± 10 minutes ;
    (iv) la centrifugation à 50 000 g ± 1 000 g à une température comprise entre 30 °C et 37 °C pendant 30 minutes ± 10 minutes.
  3. Procédé pour purifier des protéines selon la revendication 1 ou 2, comprenant en outre une étape de caractérisation par spectrométrie de masse de la fraction de protéines de l'étape (g), et/ou comprenant en outre une étape de sélection de protéines qui contiennent un domaine de protéase.
  4. Procédé pour purifier des protéines selon l'une quelconque des revendications 1 à 3, dans lequel l'échantillon biologique est choisi parmi les extraits d'organismes eucaryotes, de préférence les extraits d'animaux, mieux encore les extraits de mammifères, tels qu'un extrait de cerveau, un extrait de testicule, un extrait de poumon, et/ou dans lequel la fraction de protéines ayant une activité de tubuline carboxypeptidase comprend au moins une protéine ayant une identité de séquence d'acides aminés d'au moins 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % avec la séquence d'acides aminés choisie parmi les SEQ ID NO : 1, SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10 et SEQ ID NO : 11.
  5. Procédé pour purifier des protéines selon l'une quelconque des revendications 1 à 4, dans lequel la fraction de protéines natives ou recombinées ayant une activité de tubuline carboxypeptidase est en outre mise en contact avec des microtubules et le niveau de tyrosine (Y) isolée est mesuré, confirmant ainsi l'activité de tubuline carboxypeptidase de la fraction de protéines, dans lequel les microtubules comprennent de préférence des microtubules synthétiques et/ou des α-tubulines, avec Y C-terminale marquée.
  6. Procédé pour sélectionner un inhibiteur à base d'acide aminé ou de peptide capable d'inhiber une activité de tubuline carboxypeptidase parmi des inhibiteurs à base d'acide aminé ou de peptide candidats qui comprennent
    - un acide aminé choisi parmi Y ou F ayant un fragment réactif, de préférence choisi parmi époxysuccinyle, acyloxyméthyle, les aldéhydes et les cétones, de préférence époxysuccinyle, et mieux encore Eps-Y, ou un fragment peptidique ayant la séquence d'acides aminés choisie parmi EDY, EAY et EEY, ou
    - un fragment peptidique constitué de 2 à 20 acides aminés des acides aminés les plus C-terminaux d'une alpha-tubuline ayant la SEQ ID NO : 12 qui a ou non été chimiquement modifiée, ou
    - un fragment peptidique comprenant ou consistant en la séquence d'acides aminés choisie parmi les SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, ou
    - un fragment peptidique constitué d'entre 2 et 16 des acides aminés les plus C-terminaux de la séquence d'acides aminés Nter-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-Cter (SEQ ID NO: 20), dans laquelle
    - X1, X2, X5, X7, X9 et X13 sont des acides aminés hydrophobes, de préférence choisis parmi G, A ou V,
    - X3, X6, X8, X10, X11, X12, X14 et X15 sont des acides aminés chargés négativement, de préférence choisis parmi E ou D,
    - X4 est une chaîne latérale non chargée polaire, de préférence choisie parmi S, T, N ou Q, et
    - X16 est un gros acide aminé hydrophobe, choisi parmi Y ou F,
    ledit acide aminé ou fragment peptidique ayant, à la position C-terminale, un acide aminé choisi parmi Y ou F, dans lequel le procédé comprend :
    (a) la mise en contact de l'inhibiteur à base d'acide aminé ou de peptide candidat avec un mélange contenant à la fois une fraction de protéines natives ou recombinées ayant une activité de tubuline carboxypeptidase et des microtubules, qui comprennent de préférence des microtubules synthétiques et/ou des α-tubulines, avec Y C-terminale marquée ;
    (b) la mesure du niveau de Y isolée et/ou de microtubules détyrosinées.
  7. Procédé selon la revendication 6, dans lequel le niveau de Y isolée dans l'échantillon est comparé au niveau de Y isolée dans un échantillon témoin comprenant uniquement un extrait protéique obtenu au moyen d'un procédé pour purifier des protéines selon l'une quelconque des revendications 1 à 6 et des microtubules.
  8. Procédé selon la revendication 6 ou 7, dans lequel l'acide aminé ayant un fragment réactif est Eps-Y ou le fragment peptidique de l'inhibiteur à base de peptide candidat a la séquence d'acides aminés choisie parmi EDY, EAY et EEY, et/ou dans lequel l'inhibiteur à base de peptide candidat comprend en outre un fragment réactif, de préférence choisi parmi époxysuccinyle, acyloxyméthyle, les aldéhydes et les cétones.
  9. Inhibiteur à base d'acide aminé ou de peptide destiné à être utilisé dans le traitement d'un désordre impliquant une détyrosination des microtubules altérée chez un animal, dans lequel l'inhibiteur à base d'acide aminé ou de peptide comprend
    - un acide aminé choisi parmi Y ou F ayant un fragment réactif, de préférence choisi parmi époxysuccinyle, acyloxyméthyle, les aldéhydes et les cétones, de préférence époxysuccinyle, et mieux encore Eps-Y, ou
    - un fragment peptidique ayant la séquence d'acides aminés choisie parmi EDY, EAY et EEY, ou
    - un fragment peptidique constitué de 2 à 20 acides aminés des acides aminés les plus C-terminaux d'une alpha-tubuline ayant la SEQ ID NO : 12 qui a ou non été chimiquement modifiée, ou
    - un fragment peptidique comprenant ou consistant en la séquence d'acides aminés choisie parmi les SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, ou
    - un fragment peptidique constitué d'entre 2 et 16 des acides aminés les plus C-terminaux de la séquence d'acides aminés Nter-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-Cter (SEQ ID NO: 20), dans laquelle
    - X1, X2, X5, X7, X9 et X13 sont des acides aminés hydrophobes, de préférence choisis parmi G, A ou V,
    - X3, X6, X8, X10, X11, X12, X14 et X15 sont des acides aminés chargés négativement, de préférence choisis parmi E ou D,
    - X4 est une chaîne latérale non chargée polaire, de préférence choisie parmi S, T, N ou Q, et
    - X16 est un gros acide aminé hydrophobe, choisi parmi Y ou F,
    ledit acide aminé ou fragment peptidique ayant, à la position C-terminale, un acide aminé choisi parmi Y ou F,
    et lequel inhibiteur à base d'acide aminé ou de peptide inhibe au moins partiellement une activité de tubuline carboxypeptidase.
  10. Inhibiteur à base d'acide aminé ou de peptide destiné à être utilisé selon la revendication 9, dans lequel l'acide aminé ayant un fragment réactif est EpsY ou dans lequel le fragment peptidique de l'inhibiteur à base de peptide a une séquence d'acides aminés choisie parmi EDY, EAY et EEY, et/ou dans lequel l'inhibiteur à base de peptide comprend en outre un fragment réactif, de préférence choisi parmi époxysuccinyle, acyloxyméthyle, les aldéhydes et les cétones.
  11. Inhibiteur à base d'acide aminé ou de peptide destiné à être utilisé selon la revendication 9 ou 10, dans lequel l'inhibiteur à base d'acide aminé ou de peptide inhibe de manière irréversible ou réversible une activité de tubuline carboxypeptidase.
  12. Inhibiteur à base d'acide aminé ou de peptide destiné à être utilisé selon l'une quelconque des revendications 9 à 11, dans lequel le désordre est choisi parmi les maladies neurodégénératives, de préférence choisi parmi la maladie d'Alzheimer, la maladie de Parkinson, les troubles psychiatriques, et les troubles neuraux, les cancers, de préférence choisis parmi le cancer du côlon et le neuroblastome, les dystrophies musculaires, les maladies cardiaques, les troubles vasculaires, l'infertilité, la dégénérescence rétinienne et les ciliopathies, mieux encore choisi parmi les maladies neurodégénératives, les cancers et les dystrophies musculaires.
  13. Composition pharmaceutique comprenant une quantité efficace du point de vue thérapeutique d'un inhibiteur à base d'acide aminé ou de peptide selon l'une quelconque des revendications 9 à 11.
EP18743458.4A 2017-07-18 2018-07-18 Procédés de purification de protéines ayant une activité carboxypeptidase de tubuline et leurs inhibiteurs à base peptidique Active EP3655421B1 (fr)

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